Continuous wave (CW) spectrophotometers have been widely used to determine in vivo concentration of an optically absorbing pigment (e.g., hemoglobin, oxyhemoglobin) in biological tissue. The CW spectrophotometers, for example, in pulse oximetry introduce light into a finger or the ear lobe to measure the light attenuation and then evaluate the concentration based on the Beer Lambert equation or modified Beer Lambert absorbance equation. The Beer Lambert equation (1) describes the relationship between the concentration of an absorbent constituent (C), the extinction coefficient (.epsilon.), the photon migration pathlength &lt;L&gt;, and the attenuated light intensity (I/I.sub.o). ##EQU1## However, direct application of the Beer Lambert equation poses several problems. Since the tissue structure and physiology vary significantly, the optical pathlength of migrating photons also varies significantly and can not be simply determined from geometrical position of a source and detector. In addition, the photon migration pathlength itself is a function of the relative concentration of absorbing constituents. As a result, the pathlength through an organ with high blood hemoglobin concentration, for example, will be different from the same with a low blood hemoglobin concentration. Furthermore, the pathlength is frequently dependent upon the wavelength of the light since the absorption coefficient of many tissue constituents is wavelength dependent. One solution to this problem is to determine .epsilon., C, and &lt;L&gt; at the same time, but this is not possible with the CW oximeters.
Furthermore, for quantitative measurement of tissue of a small volume (e.g., a finger) photon escape introduces a significant error since the photons escaped from the tissue are counted as absorbed.
There are several reasons for using in vivo tissue oximetry. Although the arterial oxygen saturation can be in vitro quantified, it is not possible to estimate the change in the hemoglobin oxygen concentration as it leaves an artery and enters the capillary bed. Neither is it possible to determine the intermediate value of oxygen saturation in a particular capillary bed from the venous drainage since no technique has been devised for drawing a blood sample directly from the capillary bed.
In the time resolved (TRS-pulse) and phase modulation (PMS) spectrophotometers that can measure the average pathlength of migrating photons directly, but the proper quantitation of the time resolved or frequency resolved spectra can be performed only when the spectra are collected at a relatively large source-detector separation. This separation is difficult to achieve for a small volume of tissue such as the earlobe, a finger or a biopsy tissue.
Therefore, there is a need for a spectrophotometric system and method for quantitative examination of a relatively small volume of biological tissue.